The essential role of the thymus gland in the development of immunological competence in animals and man is now generally accepted. In thymectomized animals thymus grafts can restore immunological competence. Findings of this nature have led to the discovery of many different thymic factors. Most of these factors are derived from the thymus gland but some are obtained from serum or thymus epithelial cell cultures. For reviews and general discussion on the nature of thymic factors and how they function in the development of the immune response, see, inter alia, Tada, T. et al Adv. Immunology 28, 1 (1979); Golub, E. S. The Cellular Basis of the Immune Response Sinauer Mass. 1977, chapter 9; White, A. Ann. N.Y. Acad. Sci. 332, 23 (1979); and Kruisbeeck, A. Thymus 1 163 (1979).
The mammalian immune system consists of a sophisticated complex of interactive cells and cell products. The immune system develops in the fetus and neonatally, just after birth. Lymphocyte progenitor cells, derived from pluripotential hemopoietic stem cells, first appear in the yolk sac of the developing embryo and in the fetal liver. After birth they are found in the bone marrow, where they persist for life. These lymphocyte progenitor cells are capable of irreversibly differentiating into either of two classes of immunologically competent cells: T-lymphocytes or B lymphocytes. Both classes of lymphocytes are ubiquitous in the blood, lymph, spleen and lymph nodes. T and B lymphocytes appear to be morphologically similar--they are generally small, motile, nonphagocytic cells--yet they bear different immunogenic markers on their plasma membranes and perform distinctly differential immunological functions.
T lymphocytes (hereinafter referred to as T-cells) develop inside the thymus gland from lymphocyte progenitor cells which have migrated through the bloodstream from the bone marrow. Factors with hormone-like effect product inside the thymus apparently induce the progenitor cells to differentiate into functional T-cells with protective or regulatory capcities. Following a maturation process, the T-cells leave the thymus and enter the general blood and lymph circulation.
Some of the progenitor lymphocytes become "helper" T-cells (T.sub.H), that is, they interact with the other class of lymphocytes (B-cells) to cause them to mature, divide and become antibody-producing clones. Another subclass of T-cells produced by the thymus is the "suppressor" population (T.sub.s). These cells internally regulate the immune system so that only the type and amount of antibody or protective T-cell is produced that is needed. When this suppressor system is not regulated, certain forms of arthritis or autoimmune disease can occur. Yet another subclass of T-cells that arise in the thymus are the so-called "natural killer" T-cells (or T.sub.NK). These white cells eliminate body cells that have become infected with viruses and other microbes, and they also destroy defective cells such as cancers.
The differentiation of these distinct types of immunologically competent T-cells is controlled by an as yet incompletely understood constellation of thymic factors.
The other class of lymphocytes, called B lymphocytes or B-cells, apparently differentiate inside the bone marrow, liver and spleen from lymphocyte progenitor cells. Then these cells circulate in the bloodstream. The immunological response of mature B cells may be regulated by T-cells or T-cell products. Mature B cells synthesize and secrete immunoglobin antibodies in response to antigenic stimulation. For many antigens, B-cells require the presence of T-helper cells before they can product antibodies. The mechanism of this T-B cooperation is poorly understood, but it is recognized to be inhibited by T-suppressor cells.
The present invention encompasses a thymic factor which causes immature bone marrow cells to become suppressor T-cells. This factor is herein designated inducer of T-suppressor cells, or iT.sub.s. This invention also embraces a novel method of purifying the suppressor factor of this invention.
Mammalian bone marrow cells, when incubated with this iT.sub.s factor, become competent suppressor T-cells. It is understood that this capacity of iT.sub.s to induce the effective agents of immunosuppression can be used for therapeutic purposes. For example, human bone marrow cells can be transformed into suppressor T-cells for injection into transplant patients to cause in vivo suppression of foreign tissue rejection. It is also contemplated that pharmaceutical preparations of iT.sub.s can be prepared for direct in vivo inoculation. Naturally occurring autoimmune diseases, which have been correlated with a loss of suppressor cells, may be susceptible to treatment with iT.sub.s factor. Such autoimmune diseases include systemic lupus erythematosus, hemolytic anemia, multiple sclerosis, severe ectopic eczema, hyper-IgE syndrome, and inflammatory bowel disease. See Reinherz, E. L. et al, Immunology Today (April, 1981) pp. 69-74. Allergies are also thought to derive from defects in the suppressor T-cell system; hence it is contemplated that iT.sub.s can be of therapeutic use in controlling allergies. And, since loss of the suppressor T-cell population may correlate temporarily with the severity of other clinical diseases, it is further contemplated that iT.sub.s can be used as an adjuvant medication of general utility.
During the past decade a multiplicity of factors which exhibit hormonal activity have been isolated from the mammalian thymus gland. These thymic factors from a remarkably diverse assemblage with respect to the methodologies of their extraction, states of purification, physicochemical properties, and biological activities. Several of these thymic factors have been reported to induce some sort of suppression of the immune system: e.g., anti-thymosin, Thymopoietin I and Thymopoietin II, THF, thymosin .alpha..sub.7, FTS, and SDIP. All of these reported thymic suppressor factors have reported molecular weights that are in order of magnitude lower than those measured for applicant's iT.sub.s preparations.
Great Britain Patent Specification No. 1,195,980, published June 24, 1970 described hormone-like preparations derived from thymus gland produced at Yeshiva University, N.Y. The in vivo injection of one such preparation, called anti-thymosin, was reported to inhibit lymphoid tissue proliferation and induce a decrease in the number of blood lymphocytes. This anti-thymosin appeared to have the properties of a protein of molecular weight less than 5000.
U.S. Pat. No. 4,120,951, issued Oct. 17, 1978 to Goldstein and assigned to Sloan-Kettering Institute for Cancer Research, discloses two closely related polypeptides, designated Thymopoietin I and Thymopoietin II, from bovine thymus. The Goldstein patent states that these polypeptides can be used to inhibit the uncontrolled proliferation of thymin-responsive lymphocytes. The molecular weights of Thymopoietin I and II were reported to be around 6,000 to 7,000 daltons.
Kook et al in 1975 reported the isolation of THF, a thymic hormone of molecular weight 3220 and isoelectric point 5.66-5.90. Kook, A. I. et al, Cellular Immunology 19: 151 (1975). Shohat et al recently described the induction of T suppressor cells by in vitro treatment of lymphocytes of renal allograph recipients with THF. Shohat, B. et al, Transplantation 35 (1): 68 (1983).
Ahmed et al reported that thymosin .alpha..sub.7 acts on prothymocytes to induce suppressor cells. Ahmed, A. et al, Ann. N.Y. Acad. Sci. 332: 81 (1979). Thymosin .alpha..sub.7 has a molecular weight of 2,200 daltons and a pI of around 3.5. Low, T. L. K., et al, Ann. N.Y. Acad. Sci. 332: 32 (1979).
Facteur thymique serique (FTS) is a peptide of molecular weight close to 900 that has been isolated from both thymus tissue and normal serum. Bach, J. F., in Advances in Pharmacology and Therapeutics, Vol. 4 (Pergamon Press, 1979) p. 145. FTS is reported to activate suppressor T cells in various in vivo and in vitro systems, especially when administered at high pharmalogical dosages. It is reportedly not known whether FTS stimulates mature suppressor cells or induces a maturation of suppressor T cell precursors. Bach, J. F., in Cell Lineage, Stem Cells and Cell Determination: INSERM Symposium No. 10, p. 261 (N. LeDouarin, Ed., Elsevier/North-Holland Biomedical Press, 1979).
A spleen-derived immunosuppressive peptide (SDIP) has recently been reported to have physicochemical properties and enzymatic susceptibilities similar to those of the thymic hormone FTS, supra. When injected into sheep erythrocyte (SRBC)-sensitized mice, at the last step of differentiation of the lymphocytes, SDIP reportedly reduced the plaque-forming capacity of spleen cells from the treated animals; a similar inhibitory response was observed with FTS. Lenfant, M. et al, Immunology 48: 635 (1983).
U.S. Pat. No. 4,232,498, issued to Rule on Dec. 16, 1980 discloses and claims thymic factors prepared according to a sequential acetone fractionation process. Rule disclosed a suppressor which reportedly repressed reconstitution of the immune response in lethally irradiated thymectomized mice. No physicochemical characterization of this apparently crude thymic extract was presented.
Another suppressor factor has very recently been reported. See Kasakura, S., et al, The Journal of Immunology 130 (6): 2720 (June, 1983). The reported physical description of this factor--molecular weight of 18,000-29,000 daltons, pI of 6.2-7.3, and heat resistance only to 56.degree. C.--eliminates the possibility of it being our iT.sub.s factor.
In addition to the above-mentioned thymic suppressor factors, the administration of theophylline has also been reported to activate suppressor T cells from peripheral human blood. Shapira demonstrated a lack of suppressor T-cells in patients with acute rejection episodes (ARE) following kidney transplants. After administration of theophylline-ethyl diamine (aminophyline), the ARE in 12 of 16 patients was abrogated and suppressor T-cells reappeared in their peripheral blood. Shapria, Z. Transplantation Proceedings 14 (1): 113 (1982).
It will be noted that the thymic suppressor factors in the prior art discussed above are of comparatively lower molecular weight than iT.sub.s. For example the preparation of Example 1 exhibited a measured molecular weight of approximately 65,000 daltons. Indeed, a review of the prior art indicates that most of the thymic factors which have been categorized have molecular weights of less than 10,000 daltons. See, e.g., Goldstein, A. L. et al, in Recent Progress in Harmone Research, Vol. 37, p. 369 (Greeb, R. O., Ed., Academic Press, 1981). Furthermore, the lack of homology among those small factors which have been sequenced suggests that they are not cleavage products of a common precursor. Trainin, W., et al, Immunology Today 4 (1): 16 (1983).
The few high molecular-weight thymic factors which have been described can be distinguished from iT.sub.s by their biological activities, which tend to enhance rather than suppress immunological competence.
For example, Mizutani et al extracted two purified hypocalcemic proteins, designated TP.sub.1 and TP.sub.2, from bovine thymus gland. The molecular weight of TP.sub.1 was found to be 68,000 daltons and that of TP.sub.2 to be 57,000 daltons. Both are heat labile (56.degree. C., 30 min.). Significant increases in antibody-producing cells were found, as indicated by increases in plaque forming cells, when either TP.sub.1 or TP.sub.2 was injected into neonatal mice. Mizutani et al, Chem. Pharm. Bull. 25 (9): 2156 (1977).
White et al obtained a homogeneous protein with thymic hormone-like activity from blood serum. Its molecular weight was determined to be 56,700.+-.300 daltons, and its physical, chemical, and immunological properties indicated an identity with authentic human prealbumin. The biological activities of this high molecular weight compound included inducing an increase in the numbers of sheep erythrocyte plaque-forming cells (IgM) in vitro by spleen cells from neonatally thymectomized mice treated in vivo with the purified compound. White et al, Annals New York Academy of Sciences 332: 1 (1979). The use of human serum prealbumin for increasing immunological competence was the subject of U.S. Pat. No. 4,046,877, issued to White et al on Sept. 6, 1977 and assigned to Syntex (U.S.A.) Inc. The data presented in the patent's Example 3 indicates that human prealbumin significantly increased the capacity of spleen cells to synthesize IgM and IgG antibodies, as evidenced by increased numbers of plaque-forming cells.
Pierschbacher et al reported the extraction of a lymphocyte stimulating hormone, denoted LSHr, from beef thymus. LSHr reportedly is a peptide with a molecular weight of 80,000 and an isoelectric point of about 4.55 Administration of microgram quantities of LSHr to nude mice for 2-3 weeks induced development of T-cell function as determined by antibody response to a T-dependent antigen and a response of spleen cells to T-cell mitogen. Ann. N.Y. Acad. Sci. 332: 49 (1979).
Other high molecular-weight thymic factors have been reported by Jin et al, who reportedly extracted a mixture of polypeptides, of molecular weights 9000-68,000 daltons and isoelectric points 5.0-7.5, from pork thymus. Said factors reportedly increase rosette formation in fetal thymocytes, which indicates generally the induction of T-cell differentiation. Jin, T. et al J. Nanking University 1: 115 (1979), cited in Goldstein et al, Recent Progress in Hormone Research, Vol. 37, supra, at p. 381.
The methods employed to extract the above-mentioned factors from thymus tissue typically consist of two general steps. First, a crude extract is prepared from thymus gland--typically by homogenization, heat treatment, centrifugation and filtration. Second, the factor or factors in the crude extract are isolated by enrichment procedures--typically by dialysis, molecular sieve chromatography, affinity chromatography, and/or preparative electrophoresis.
The extraction procedure ofthe present invention, as will be discussed in greater detail below, also involves a two-part procedure. A crude thymus extract is first prepared in conformance with procedures well known in the art. Said crude extract is then subjected to a novel and surprisingly effective enrichment protocol which for the first time has permitted the isolation of a stable, high molecular-weight T-suppressor factor from mammalian thymus glands.
The crude thymic extract, as used as the start of my inventive process, is prepared in general conformity with the methods described by others; see, e.g., A. Goldstein's Thymic Fraction 2, in U.S. Pat. No. 4,010,148. I then fractionate the crude thymic extract by molecular sieve chromatography and remove contaminants by affinity chromatography using immunoadsorbents.